Biomedical research has evolved significantly over the past several years, with the large-scale screening of whole genomes complementing focused studies on a few genes or proteins. This evolution has encompassed applications ranging from functional analysis of unknown genes to identification of disease-related genes, screening in drug discovery and clinical diagnostics. There has been a concurrent surge in technology development to facilitate large-scale biological analysis. In general, these technologies have two components, the assay chemistry and the detection platform. Perhaps the best publicized detection platform of recent years is the flat microarray. Configured as “DNA chips,” these flat microarrays offer the promise of whole genome analysis of single samples. Each element or “spot” on a flat surface array contains a target-specific receptor, for example a DNA molecule to detect a specific DNA sequence, and the signal originating from that element reports the presence of a target molecule. A related detection platform that is proving to be compatible with a range of assay chemistries in a high-throughput format is the use of encoded microparticle in combination with flow-based analysis cytometry, also known as Suspension Array Technology (SAT).
Suspension array technology employs fluorescence-encoded microspheres as array elements that bear specific receptor molecules. In SAT, microspheres having distinct optical properties, for example light scatter or fluorescence from an internal dye, are employed as solid supports for a variety of molecular analyses. By careful adjustment of these intrinsic optical properties, it is possible to prepare arrays of microspheres in which individual microsphere subsets can be identified and used to perform multiplexed analysis. Conceptually, microsphere arrays are similar to flat-surface microarrays, with distinct quanta of an intrinsic optical parameters substituting for physical location on a surface. While fluorescent- or optically-encoded microspheres have improved the flexibility of array-based analysis, that approach faces limitations in both the preparation and use of the microspheres. For example, the ability to reproducibly dye microspheres is problematic such that lot-to-lot variations in microparticles can be a problem. Moreover, the use of fluorescent dyes to encode the microparticles limits the number of analytical measurements, which also employ fluorescence detection, that can be made. An encoding method that did not require, but that was compatible with, fluorescence detection would be desirable.
The analysis of single nucleotide polymorphisms (SNPs), provides a useful example of the types of analysis that can be performed. The human genome project has shown that the DNA sequence from any two individuals is about 99.9% identical, and that the phenotypic differences between individuals are conferred largely by the 0.1% of the sequence that is different. The vast majority of this sequence variation is in the form of single nucleotide polymorphisms (or SNPs), sites in the genome where a single base varies between chromosomes in the same individual or between different individuals.
As genetic markers, SNPs have great potential for use in disease diagnostics and the discovery of new drugs. Major pharmaceutical companies and academic genome centers are involved in a major effort to discover and map SNPs. Unfortunately, conventional methods of genotyping are too slow and expensive to allow this new data to be applied on a large scale.
High throughput methods have been developed for large scale SNP scoring based on single base extension (SBE) of oligonucleotide primers using arrays of fluorescently labeled microspheres. Such systems provide accurate genotyping in a flexible format with ten-fold higher throughput and ten-fold lower costs than conventional genotyping methods. For example, U.S. Pat. No. 5,981,180 by Chandler et al. describes a method for the multiplexed diagnostic and genetic analysis of enzymes, DNA fragments, antibodies and other biomolecules. In their method, an appropriately labeled beadset is constructed, the beadset is exposed to a clinical sample, and the combined beadset/sample is analyzed by flow cytometry. Their method employs a pool of beadsets wherein beads within a subset differ in at least one distinguishing characteristic from beads in any other beadset. In that manner, the subset to which a bead belongs can be readily determined after beads from different subsets are combined. The distinguishing characteristics between beadsets are provided by incorporation of two or more fluorophores into the beads. Given suitable fluorophores and detection equipment, use of multiple fluorophores could expand the multiplexing power of the system.
However, the multiplexed analysis capacity of typical fluorescent microsphere arrays is currently limited to one hundred simultaneous assays, and expansion beyond this number involves a number of technical challenges. In addition, the routine preparation of these fluorescent microspheres still presents problems.
Solid phase arrays have also been used for the rapid and specific detection of multiple polymorphic nucleotides. Typically, an allele-specific hybridization probe is linked to a solid support and a target nucleic acid (e.g., a genomic nucleic acid, an amplicon, or, most commonly, an amplified mixture) is hybridized to the probe. Either the probe, or the target, or both, can be labeled, typically with a fluorophore. Where the target is labeled, hybridization is detected by detecting bound fluorescence. Where the probe is labeled, hybridization is typically detected by quenching of the label. Where both the probe and the target are labeled, detection of hybridization is typically performed through monitoring of a color shift resulting from proximity of the two bound labels. A variety of labeling strategies, labels, and the like, particularly for fluorescent based applications are described.
In one embodiment, an array of probes is synthesized on a solid support. Exemplary solid supports include glass, plastics, polymers, metals, metalloids, ceramics, organics, and the like. Using chip masking technologies and photoprotective chemistry it is possible to generate ordered arrays of nucleic acid probes. These arrays, which are known, e.g., as “DNA chips,” or as very large scale immobilized polymer arrays (VLSIPS™ arrays) can include millions of defined probe regions on a substrate having an area of about 1 cm2 to several cm2, thereby incorporating sets of from a few to millions of probes. The construction and use of solid phase nucleic acid arrays to detect target nucleic acids is well described in the literature. See, e.g., Fodor et al. (1991) Science, 251: 767-777; Hubbell U.S. Pat. No. 5,571,639; and, Pinkel et al. PCT/US95/16155 (WO 96/17958).
Magnetic particles made from magnetite and inert matrix materials have long been used in the field of biochemistry. Such particles generally range in size from a few nanometers up to a few microns in diameter and may contain from 15% to 100% magnetite. They are often described as superparamagnetic particles or, in the larger size range, as beads. The usual methodology is to coat the surface of the particles with some biologically active material that will cause them to bond strongly with specific chemical species, microscopic objects or particles of interest (e.g., proteins, viruses, and DNA fragments). The particles then become “handles” by which the objects can be moved or immobilized using a magnetic gradient, usually provided by a strong permanent magnet. U.S. Pat. No. 4,537,861 by Elings et al. describes an example of tagging using magnetic particles. Specially constructed fixtures using rare-earth magnets and iron pole pieces are commercially available for this purpose. However, in this process, magnetic particles are never used in labeled subsets of particles allowing for a multiplexed assay of a sample.
In another approach using magnetic particles, U.S. Pat. No. 5,252,493 by Fujiwkara et al. describes a ultra-sensitive laser magnetic immunoassay method including: labeling an antigen or antibody with micro-particles of a magnetic substance to form a magnetic-labeled body; subjecting a specimen and the magnetic-labeled body to an antigen-antibody reaction to form a reacted body-specimen complex; separating and removing unreacted body from the reacted complex; guiding and concentrating the reacted complex magnetically; irradiating the concentrated complex with a laser beam; detecting outgoing light from a measurement system to provide a quantitative result in the picogram range. Again in this process, magnetic particles are never used in labeled subsets of particles allowing for a multiplexed assay of a sample.
It would be beneficial if another method were available for detecting the presence of a sought-after, predetermined target, e.g., such as a nucleotide sequence or allelic variants. It would further be beneficial if such a detection method were capable of providing multiple analyses in a single assay (multiplex assays).